Radiolabeled sugars for imaging of fungal infections

ABSTRACT

Disclosed herein are compounds having a structure according to Formula I and optionally Formula IV. 
     
       
         
         
             
             
         
       
     
     The compounds may be radiolabeled compounds useful for diagnosis and/or imaging fungal infections. In such embodiments, at least one substituent is a radionuclide, such as  18 F. Also disclosed are precursor compounds according to Formula I and/or IV that are useful for making the radiolabeled compounds. In such embodiments, the precursor compound comprises at least one leaving group suitable for introducing a radionuclide, such as  18 F, at a desired position. 
     Also disclosed are methods for making and using the compounds, including embodiments of a method for imaging and/or diagnosing a fungal infection in a subject.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the earlier filing date of U.S.provisional patent application No. 62/882,023, filed Aug. 2, 2019, whichis incorporated herein by reference in its entirety.

FIELD

Provided herein are radiolabeled sugars, such as [¹⁸F]-rhamnose and[¹⁸F]-cellobiose, and methods of their use to detect and monitor afungal infection.

BACKGROUND

Fungal infections remain a major health burden with very high mortalityand morbidity in immunosuppressed cancer and stem cell transplantpatients, in advanced HIV disease and in some congenitalimmunodeficiencies. A recent report estimated global mortality fromfungal disease to be >1.6 million, similar to that of tuberculosisand >3-fold that of malaria (Bongomin et al., J Fungi (Basel), 3, 2017).Yet, despite the magnitude of the problem, there are currently noclinically-available fungal-specific imaging agents. FDG PET is anonspecific technique that can be used for imaging patients withsuspected active fungal infections. It cannot however, differentiatebetween infection and inflammation, between the various fungal pathogensor differentiate fungi from other pathogens such as bacteria. Thedevelopment of fungus-specific imaging has been attempted using varyingapproaches including targeted antibodies (Rolle et al., Proc Natl AcadSci U S A, 113:E1026-3, 2016), 99mTc labeled MORF oligomers targetingfungal ribosomal RNA (rRNA) (Wang et al., Nucl Med Biol, 40:89-96,2013), and the use of radiolabeled siderophores (Haas et al., PLoSPathog, 11: e1004568, 2015). Many of those ligands however are still inearly stages of development, have been abandoned or mostly, have notbeen tested in humans. Thus, new fungal-specific imaging ligands areneeded.

SUMMARY

Provided herein are new fungal-specific imaging ligands, which exploitmetabolic pathways that are selectively expressed by fungi, but not bymammalian cells or bacteria. These fungal-specific imaging ligands canbe used to diagnose and/or monitor a fungal infection in vivo, withoutthe need for invasive procedures or biopsies.

Disclosed herein are compounds having a formula I

With respect to Formula I, R¹ is a radionuclide, OH, OR⁶, OR⁷ or X. X is

where each of R⁷, R⁸, R⁹ and R¹⁹ independently is a radionuclide, OH,OR⁶, or OR⁷. Each of R², R³ and R⁴ independently is OH, OR⁶, OR⁷ or aradionuclide. R⁵ is H, OH, OR⁶, OR⁷ or a radionuclide, with theprovision that when R¹ is X then R⁵ is OH, OR⁶, OR⁷ or a radionuclide,and when R¹ is other than X, then R⁵ is H, OR⁷, or a radionuclide. R⁶ isacetyl, formyl, methoxyacetyl, benzoyl, haloacetyl or trialkylsilyl, andin some embodiments, R⁶ is acetyl. And R⁷ is triflate, mesylate ortosylate, and in some embodiments, R⁷ is triflate.

Also with respect to Formula I, one of the following conditions (a) or(b) applies:

(a) if R¹ is X then either at least one of R²-R⁵ and R⁸-R¹¹ is aradionuclide and the rest are OH, or at least one of R²-R⁵ and R⁸-R¹¹ isOR⁷ and the rest are OR⁶; and

(b) if R¹ is other than X, then at least one of R¹-R⁵ and R⁸-R¹¹ is aradionuclide and the rest are OH except for R⁵ which is either aradionuclide or H, or at least one of R¹-R⁵ and R⁸-R¹¹ is OR⁷ and therest are OR⁶ except for R⁵ which is either OR⁷ or H.

In any embodiments, the radionuclide may be ¹⁸F.

In some embodiments, R¹ is ¹⁸F, OH, OR⁶, or OR⁷ and condition (b)applies. In such embodiments, each of R¹, R², R³, and R⁴ independentlymay be ¹⁸F or OH; R⁵ may be ¹⁸F or H; and at least one of R¹-R⁵ may be¹⁸F. And in some embodiments, R¹ is ¹⁸F, R²-R⁴ are OH, and R⁵ is H; R²is ¹⁸F, R¹, R³ and R⁴ are OH, and R⁵ is H; R³ is ¹⁸F, R¹, R² and R⁴ areOH, and R⁵ is H; R⁴ is ¹⁸F, R¹, R² and R³ are OH, and R⁵ is H; or R⁵ is¹⁸F, and R¹-R⁴ are OH. Additionally, or alternatively, the compound mayhave a Formula II

And with respect to Formula II, the compound may be

In other embodiments, each of R¹, R², R³, and R⁴ independently is OR⁶ orOR⁷; R⁵ is OR⁷ or H; and at least one of R¹-R⁵ is OR⁷. In some suchembodiments, one of R¹-R⁵ is OR⁷ and the rest are OR⁶. And/or in someembodiments, the compound has a formula selected from

Additionally, in some embodiments, R¹ is OR⁷, R²-R⁴ are OR⁶, and R⁵ isH; R² is OR⁷, R¹, R³ and R⁴ are OR⁶, and R⁵ is H; R³ is OR⁷, R¹, R² andR⁴ are OR⁶, and R⁵ is H; R⁴ is OR⁷, R¹, R² and R³ are OR⁶, and R⁵ is H;or R⁵ is OR⁷, and R¹-R⁴ are OR⁶. And R⁶ may be acetyl and R⁷ may betriflate.

In other embodiments of Formula I, the compound has a structureaccording to Formula IV and condition (a) applies

In some such embodiments, each of R²-R⁵ and R⁸-R¹¹ independently is ¹⁸For OH, and at least one of R²-R⁵ and R⁸-R¹¹ is ¹⁸F, and in certainembodiments, one of R²-R⁵ and R⁸-R¹¹ is ¹⁸F and the rest of R²-R⁵ andR⁸-R¹¹ are OH.

Additionally or alternatively, the compound may have a structureaccording to Formula V

With respect to Formula V, in some embodiments, R² is ¹⁸F and R³-R⁵ andR⁸-R″ are OH; R³ is ¹⁸F and R², R⁴, R⁵ and R⁸-R¹¹ are OH; R⁴ is ¹⁸F andR², R³, R⁵ and R⁸-R¹¹ are OH; R⁵ is ¹⁸F and R²-R⁴ and R⁸-R¹¹ are OH; R⁸is ¹⁸F and R²-R⁵ and R⁹-R¹¹ are OH; R⁹ is ¹⁸F and R²-R⁵ and R⁸, R¹⁰ andR¹¹ are OH; R¹⁰ is ¹⁸F and R²-R⁵ and R⁸, R⁹ and R¹¹ are OH; or R¹¹ is¹⁸F and R²-R⁵ and R⁸ R¹⁰ are OH. In certain embodiments, R⁹ is ¹⁸F andR²-R⁵ and R⁸, R¹⁰ and R¹¹ are OH, but in other embodiments, R² is ¹⁸Fand R³-R⁵ and R⁸-R¹¹ are OH.

In other embodiments of Formula IV, each of R²-R⁵ and R⁸-R¹¹independently is OR⁶ or OR⁷, and at least one of R²-R⁵ and R⁸-R¹¹ isOR⁷. In some embodiments, one of R²-R⁵ and R⁸-R¹¹ is OR⁷ and the rest ofR²-R⁵ and R⁸-R¹¹ are OR⁶. And in certain embodiments, R² is OR⁷ andR³-R⁵ and R⁸-R¹¹ are OR⁶; R³ is OR⁷ and R², R⁴, R⁵ and R⁸-R¹¹ are OR⁶;R⁴ is OR⁷ and R², R³, R⁵ and R⁸-R¹¹ are OR⁶; R⁵ is OR⁷ and R²-R⁴ andR⁸-R¹¹ are OR⁶; R⁸ is OR⁷ and R²-R⁵ and R⁹-R¹¹ are OR⁶; R⁹ is OR⁷ andR²-R⁵ and R⁸, R¹⁰ and R¹¹ are OR⁶; R¹⁰ is OR⁷ and R²-R⁵ and R⁸, R⁹ andR¹¹ are OR⁶; or R¹¹ is OR⁷ and R²-R⁵ and R⁸-R¹⁰ are OR⁶. And R⁶ may beacetyl and R⁷ may be triflate.

Also provided are compositions that include one or more radiolabeledcompounds and a pharmaceutically acceptable carrier, such as water orsaline.

Also provided are methods of using the disclosed radiolabeled compoundsto detect a fungus, in vivo to diagnose and/or monitor a fungalinfection in a subject.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. in vitro uptake of ³H-2-deoxygluose (2-DG) and ³H-rhamnosein (FIG. 1A) A. fumigatus (FIG. 1B) E. coli (FIG. 1C) S. aureus and(FIG. 1D) J774 macrophages at various time-points. The net uptake oflive cultures are plotted in the graphs after subtracting the backgrounduptake by heat-killed cultures (except J774 macrophages where onlyuptake in live cultures was measured). (FIG. 1E) In vitro uptake of¹⁸F-rhamnose in live and heat killed A. fumigatus and E. coli. (FIG. 1F)Representative autoradiography images of in vivo uptake of ³H-rhamnosein the lungs of healthy, poly (I:C) treated (sterile inflammation) andA. fumigatus infected mice is shown in the top panels. Bright fieldimages of the respective lung sections are in the bottom panel.

FIG. 2 is a bar graph showing ³H-L-Rhamnose results from biodistributionstudies showing increased uptake in the lungs of infected compared tocontrol mice and mice with sterile lung inflammation.

FIGS. 3A-3B. (FIG. 3A) Representative dynamic [¹⁸F]-rhamnose PET images,averaged from 520-3520 seconds post injection. Increased uptake is seenin the lungs of nasopharyngeally-infected pulmonary IA (AF NP) micewhile no appreciable uptake is seen in the lungs of control or sterilelung (poly (I:C)inflammation mice. The first 520 seconds were removedfrom analysis to reduce potential effects of increased vascularity afterinjection. (FIG. 3B) Time activity curve of [¹⁸F]-rhamnose uptake incontrol, poly (I:C), and AF NP models from 0-3370 seconds post[¹⁸F]-rhamnose injection.

FIGS. 4A-4C. In vitro uptake of ³H-cellobiose by Aspergillus (FIG. 4A)but not by macrophage (J744) cell lines (FIG. 4B). (FIG. 4C)Biodistribution studies show increased activity in the lungs of infectedmice compared to control animals Increased activity in the brain mayreflect free labeled glucose following hydrolysis of cellobiose withsecondary uptake by the brain.

FIGS. 5A-5B: Autoradiography and GMS staining of (FIG. 5A) lung in anAspergillus fumigatus nasopharyngeally-infected mouse and (FIG. 5B)brain in an Aspergillus fumigatus IV infected mouse. GMS stainingconfirms the presence of fungal hyphae with corresponding 3H-Cellobioseuptake

FIG. 6: ¹⁸F-deoxycellobiose with the isotope located on C2 of the firstglucose molecule or on the C2 of the second glucose molecule.

DETAILED DESCRIPTION I. Terms

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

When chemical structures are depicted or described, unless explicitlystated otherwise, all carbons are assumed to include implicit hydrogenssuch that each carbon conforms to a valence of four. For example, in thestructure on the left-hand side of the schematic below there are ninehydrogen atoms implied. The nine hydrogen atoms are depicted in theright-hand structure.

Sometimes a particular atom in a structure is described in textualformula as having a hydrogen or hydrogen atoms, for example —CH₂CH₂—. Itwill be understood by a person of ordinary skill in the art that theaforementioned descriptive techniques are common in the chemical arts toprovide brevity and simplicity to description of organic structures.

Administration: To provide or give a subject an agent, such as aradiolabeled sugar provided herein and/or an anti-fungal agent, by anyeffective route. Exemplary routes of administration include, but are notlimited to, topical, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, intraosseous, intra-arterial, andintravenous), oral, ocular, sublingual, rectal, transdermal, intranasal,vaginal and inhalation routes.

Alkyl: A saturated aliphatic hydrocarbyl group having from 1 to 25(C₁₋₂₅) or more carbon atoms, more typically 1 to 10 (C₁₋₁₀) carbonatoms such as 1 to 6 (C₁₋₆) carbon atoms or 1 to 4 (C₁₋₄) carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃), ethyl (—CH₂CH₃), n-propyl (—CH₂CH₂CH₃),isopropyl (—CH(CH₃)₂), n-butyl (—CH₂CH₂CH₂CH₃), isobutyl(—CH₂CH₂(CH₃)₂), sec-butyl (—CH(CH₃)(CH₂CH₃), or t-butyl (—C(CH₃)₃).

Contact: Placement in direct physical association, including a solid ora liquid form. Contacting can occur in vitro or ex vivo, for example, byadding a reagent to a sample, or in vivo by administering to a subject.

Detect or measure: To determine if a particular agent (e.g., fungalinfection, radiolabeled sugar provided herein) is present or absent, andin some examples further includes semi-quantification or quantificationof the agent if detected.

Effective amount: An amount of a composition that alone, or togetherwith an additional therapeutic agent(s) sufficient to achieve a desiredeffect, for example in vivo The effective amount of the agent (such asan anti-fungal agent) can be dependent on several factors, including,but not limited to the subject being treated (e.g., whether the subjectis immune compromised), the severity, stage, and type of fungalinfection being treated, the particular therapeutic agent, and themanner of administration. Effective amounts also can be determinedthrough various in vitro, in vivo or in situ immunoassays. One or moreanti-fungal agents can be administered in a single dose, or in severaldoses, as needed to obtain the desired response.

In one example, an effective amount or concentration is one that issufficient to treat a fungal infection in a subject, for example byreducing or inhibiting one or more symptoms associated with theinfection. The infection and symptoms need not be completely eliminatedfor the method to be effective. For example, administering one or moreanti-fungal agents to a subject can substantially decrease the fungalinfection (or one or more signs or symptoms of the infection) in thesubject, such as a decrease of at least 20%, at least 50%, at least 80%,at least 90%, at least 95%, at least 98%, or even at least 100%, ascompared to the amount present prior to administration of the ng one ormore anti-fungal agents.

Fungus: A member of the group of eukaryotic organisms that includeschitin in their cell walls. Includes fungal organisms that can infect asubject, such as mammals and birds. Fungal infections invade one or moretissues causing infection, for example in the skin or internal organssuch as the blood, kidney, heart, esophagus, lungs, sinuses,gastrointestinal tract, and central nervous system (e.g., brain, spinalcord). Exemplary fungi that can be diagnosed or treated using themethods provided herein include, but are not limited to, Aspergillus(which can cause Aspergillosis), such as A. fumigatus, A. flavus, A.terreus, and A. niger; Candida (which can cause candidiasis), such as C.albicans; Cryptococcus (which can cause Cryptococcosis), such as C.neoformans and C. gattii; and Mucormycetes (which can causemucormycosis, sometimes called zygomycosis), such as Rhizopus species,Mucor species, Rhizomucor species, Syncephalastrum species,Cunninghamella bertholletiae, Apophysomyces species, and Lichtheimia(formerly Absidia).

Halo: Fluoro, chloro, bromo or iodo.

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds, such as one or more radiolabeled compoundsprovided herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Precursor and precursor compound: Compounds that are used to make aradiolabeled compound, but typically do not comprise a radionuclidethemselves. A person of ordinary skill in the art understands thatbecause transport of radioactive compounds may be problematic, due totransport restrictions and that fact that the radioactive isotope decaysover time, the precursor compound may be prepared, stored and/ortransported, and the radionuclide is added prior to use, such as by anend user. Typically, a precursor compound comprises a leaving group thatcan exchanged or displaced when the radionuclide is introduced. Theprecursor compound also may comprise one or more protecting groups thatprotect other functional groups when the radionuclide is introduced, andcan be removed prior to use.

Radiolabeled: A compound that comprises a radionuclide.

Radionuclide: A radioactive isotope. For example, ¹⁸F is a radionuclideof fluorine.

Sample: A sample of biological material obtained from a subject, whichcan include cells (such as fungal cells), proteins, nucleic acidmolecules (such as DNA and/or RNA). Biological samples include allclinical samples useful for detection of disease, such as a fungalinfection, in subjects. Appropriate samples include any conventionalbiological samples, including clinical samples obtained from a human orveterinary subject. Exemplary samples include, without limitation,cells, cell lysates, blood smears, cytocentrifuge preparations, cytologysmears, bodily fluids (e.g., blood, plasma, serum, stool/feces, saliva,sputum, urine, bronchoalveolar lavage, cerebrospinal fluid (CSF), nasalswabs, etc.), or fine-needle aspirates. Samples may be used directlyfrom a subject, or may be processed before analysis (such asconcentrated, diluted, purified). In a particular example, a sample orbiological sample is obtained from a subject having, suspected ofhaving, or at risk of having a fungal infection.

Subject or patient: A term that includes human and non-human mammals. Inone example, the subject is a human or veterinary subject, such as amouse, non-human primate, cow, pig, rabbit, rat, horse, cat, dog, andthe like. In some examples, the subject is a mammal (such as a human)who has a fungal infection, or is being treated for a fungal infection.In some examples, the subject is immune compromised. In some examples,the subject is immune compromised and has a fungal infection, such asinvasive aspergillosis.

In some examples, a subject analyzed with the disclosed methods is onewho has received a transplant (e.g., transplant of at least one of astem cell or solid organ, such as lung, heart, liver, kidney, pancreas,or intestine). In some examples, a subject analyzed with the disclosedmethods is one who has a primary immunodeficiency (examples of primaryimmunodeficiency diseases include those listed in Al-Herz et al.(Frontiers in Immunology, volume 5, article 162, Apr. 22, 2014, hereinincorporated by reference in its entirety), e.g., T-B+SCID, T-B−SCID,WHIM syndrome, IL-7 receptor severe combined immune deficiency (SCID),Adenosine deaminase deficiency (ADA) SCID, Purine nucleosidephosphorylase (PNP) deficiency, Wiskott-Aldrich syndrome (WAS), Chronicgranulomatous disease (CGD), Leukocyte adhesion deficiency (LAD),Duchenne muscular dystrophy, Glycogen storage disease type IA, RetinalDystrophy, and X-linked immunodeficiency with magnesium defect,Epstein-Barr virus infection, and neoplasia (XMEN)). In some examples, asubject analyzed with the disclosed methods is one who has HIV or AIDS.In some examples, a subject analyzed with the disclosed methods is onewho has cancer, such as a cancer of the lung, liver, pancreas, breast,prostate, ovary, colon, rectum, head and neck, brain, bone, or blood.

The terms “¹⁸F-rhamnose” and “[¹⁸]-rhamnose” include ¹⁸F-labeledrhamnose and deoxyrhamnose analogs, such as, but not limited to,6-¹⁸F-rhamnose and ¹⁸F-deoxyrhamnose analogs where the ¹⁸F is at the 1,2, 3, 4, or 5 position.

The term “¹⁸F-cellobiose” and “[¹⁸F]-cellobiose” include ¹⁸F-labeledcellobiose and deoxycellobiose analogs, such as, but not limited to,6-¹⁸F-cellobiose, 12-¹⁸F-cellobiose, and ¹⁸F-deoxycellobiose analogswhere the ¹⁸F is at the 1, 2, 3, 8, 9, or 10 positions.

II. Overview

The inventors identified sugars and other molecules involved in themetabolic pathways of clinically-relevant fungal infections, andgenerated radiolabeled versions (3H or 14C) that were tested for invitro uptake in bacteria (gram-negative, gram-positive, Pseudomonasaeruginosa), macrophages (J774 cell line) and m clinically-relevantfungal strains including Aspergillus, Rhizomucor, Cryptococcus andCandida albicans. Organisms and cells were exposed to the radiolabeledcompounds and the in vitro uptake was measured (retained radioactivityafter incubation and washing of the cultures measured using a betacounter). If positive uptake in the fungi was observed with no or onlylow uptake in bacteria and mammalian cells, organ biodistributionstudies and autoradiography were used to determine specific uptake indifferent animal models of fungal infection. For the biodistribution andautoradiography studies showing specific uptake of the radioactivemolecules by fungal-infected animals (i.e., retained radioactivity inthe lungs in the infected animals but not in the control animals), theligand(s) specific for each type of fungi was radiolabeled with 18F orother PET isotopes. Using the radiolabeled ligand(s), live PET imagingis performed on infected mice using a microPET/CT scanner. This is doneby injecting the radioactive ligand intravenously through the tail veinand then positioning the animals inside the microPET/CT gantry andobtaining CT scan images as well as PET emission data. The images arereconstructed and analyzed using special software. To confirmspecificity for fungal infection, in vivo uptake of the ligand(s) inanimal models of sterile inflammation, as well as models of bacterialinfection (gram positive and gram negative bacteria) are performed.

Three main mouse models were developed: (1) Aspergillus lung infection(nasopharyngeal administration of 30 μl suspension of Aspergillusconidia), (2) Aspergillus hematogenous spread (intravenousadministration of 100 μl of fungal suspension via the tail vein) and (3)sterile lung inflammation model (induced by nasopharyngealadministration of 50-100 μg of poly (I:C) suspension in 30 μl of sterilePBS; poly(I:C) is a synthetic double stranded RNA which activatesToll-like receptors-3 (TLR3), thereby inducing signaling via multipleinflammatory pathways).

Additional mouse models included Gram negative (E. coli) and grampositive (S. aureus) bacterial infection models (myositis induced byinjection of 107-109 CFU of E. coli or Staphylococcus aureusintramuscularly into the caudal thigh region (hind limb)), a model ofsubcutaneous Aspergillus infection (200 μl suspension containing 5×105to 5×107 conidia injected subcutaneously, in the right dorsum) and amodel of contralateral sterile inflammation (heat killed conidia+complete Freund's adjuvant (CFA)).

Other fungal mouse models besides Aspergillus include Candida albicans(intravenous injection of 100-150 μl of Candida culture containing a CFUof 103-109) and Cryptococcus neoformans (30 μl suspension of fungalcells administered through the posterior pharyngeal method) infectedmodels.

Two sugars, L-rhamnose and cellobiose were radiolabeled and can be usedas PET ligands.

III. Compounds

Disclosed herein are radiolabeled compounds and precursors thereof.Radiolabeled compounds comprise a radionuclide, for example ¹⁸F. Theradiolabeled compounds are useful as for diagnosing certain infections,such as fungal infections, in a patient. In some embodiments, thecompounds are analogs and/or derivatives of sugars that are metabolizedby fungi, and may be selectively metabolized by the fungi, such that thepatient does not substantially metabolize the sugar. This results in theradiolabeled metabolites selectively accumulating in the fungi, therebyidentifying the fungal infection.

In some embodiments, the compound has a formula I

With respect to Formula I:

R¹ is a radionuclide, OH, OR⁶, OR⁷ or X;

X is

where each of R⁷, R⁸, R⁹ and R¹⁰ independently is a radionuclide, OH,OR⁶, or OR⁷;

each of R², R³ and R⁴ independently is OH, OR⁶, OR⁷ or a radionuclide;

R⁵ is H, OH, OR⁶, OR⁷ or a radionuclide, with the proviso that when R¹is X then R⁵ is OH, OR⁶, OR⁷ or a radionuclide, and when R¹ is otherthan X, (i.e., R¹ is a radionuclide, OH, OR⁶, OR⁷) then R⁵ is H, OR⁷, ora radionuclide;

R⁶ is a suitable protecting group, and may be an ester or silylprotecting group, such as acetyl (CH₃C(═O)—; Ac), formyl, methoxyacetyl,benzoyl, haloacetyl (such as trifluoroacetyl, chloroacetyl,dichloroacetyl, or trichloroacetyl) or trialkylsilyl (such as trimethylsilyl or triethyl silyl), and in certain embodiments, R⁶ is acetyl;

R⁷ is a suitable leaving group, such as triflate (CF₃SO_(2;) Tr),mesylate (CH₃SO₂) or tosylate (CH₃PHSO₂), and in certain embodiments, R⁷is triflate;

Also, with respect to Formula I the following conditions apply:

(a) if R¹ is X then either at least one of R²-R⁵ and R⁸-R¹¹ is aradionuclide and the rest are OH, or at least one of R²-R⁵ and R⁸-R¹¹ isOR⁷ and the rest are OR⁶; and

(b) if R¹ is other than X, then at least one of R¹-R⁵ and R⁸-R¹¹ is aradionuclide and the rest are OH except for R⁵ which is either aradionuclide or H; or at least one of R¹-R⁵ and R⁸-R¹¹ is OR⁷ and therest are OR⁶ except for R⁵ which is either OR⁷ or H.

In some embodiments, R¹ is OR⁶, OR⁷, X; each R²-R⁴ independently is OR⁶or OR⁷; R⁵ is H, OR⁶ or OR⁷; and, if present, each of R⁷, R⁸, R⁹ and R¹⁰independently is OR⁶ or OR⁷.

In other embodiments, R¹ is OH, a radionuclide, or X; each R²-R⁴independently is OH or a radionuclide; R⁵ is H, OH or a radionuclide;and, if present, each of R⁷, R⁸, R⁹ and R¹⁰ independently is OH or aradionuclide.

In any embodiments, the radionuclide may be ¹⁸F.

I. Rhamnose analogs

In some embodiments of Formula I, the compound is a rhamnose analog. Insuch embodiments, R¹ is other than X, i.e., R¹ is a radionuclide, OH,OR⁶, or OR⁷ and condition (b) applies. In any embodiments, the rhamnoseanalog may be an L-Rhamnose analog.

i) radiolabeled rhamnose

In some embodiments, the rhamnose analog is a radiolabeled analog, suchas an ¹⁸F radiolabeled rhamnose or deoxyrhamnose analog. In suchembodiments, each of R¹, R², R³, and R⁴ independently is ¹⁸F or OH, andR⁵ is ¹⁸F or H, where at least one of R¹-R⁵ is ¹⁸F. In some embodiments,one of R¹-R⁵ is ¹⁸F.

In some embodiments, the compound is ¹⁸F radiolabeled L-Rhamnose analogand has a structure according to Formula II:

With respect to Formula II, R¹-R⁴ are ¹⁸F or OH and R⁵ is ¹⁸F or H,where at least one, and optionally exactly one of R¹-R⁵ is ¹⁸F.

In an embodiment of Formulas I or II, R¹ is ¹⁸F, R²-R⁴ are OH, and R⁵ isH.

In an embodiment of Formulas I or II, R² is ¹⁸F, R¹, R³ and R⁴ are OH,and R⁵ is H.

In an embodiment of Formulas I or II, R³ is ¹⁸F, R¹, R² and R⁴ are OH,and R⁵ is H.

In an embodiment of Formulas I or II, R⁴ is ¹⁸F, R¹, R² and R³ are OH,and R⁵ is H.

In an embodiment of Formulas I or II, R⁵ is ¹⁸F, and R¹-R⁴ are OH.

In a particular embodiment of Formula II, R² is ¹⁸F, R¹, R³ and R⁴ areOH, and R⁵ is H; or R³ is ¹⁸F, R¹, R² and R⁴ are OH, and R⁵ is H; or R⁵is ¹⁸F, and R¹-R⁴ are OH.

Exemplary Rhamnose analogs according to Formula II:

ii) Radiolabeled rhamnose precursor

In other embodiments of Formula I, the compound is a precursor compoundto a radiolabeled rhamnose analog. In such embodiments, each of R¹, R²,R³, and R⁴ independently is OR⁶ or OR⁷, and R⁵ is OR⁷ or H, where atleast one of R¹-R⁵ is OR⁷, and R⁶ and R⁷ are as previously defined. Insome embodiments, exactly one of R¹-R⁵ is OR⁷, and the rest are OR⁶.

The Rhamnose precursor compound may have a structure according to anyone of Formulas III-a to III-e:

In an embodiment of Formulas I or III-a to III-e, R¹ is OR⁷, R²-R⁴ areOR⁶, and R⁵ is H.

In an embodiment of Formulas I or III-a to III-e, R² is OR⁷, R¹, R³ andR⁴ are OR⁶, and R⁵ is H.

In an embodiment of Formulas I or III-a to III-e, R³ is OR⁷, R¹, R² andR⁴ are OR⁶, and R⁵ is H.

In an embodiment of Formulas I or III-a to III-e, R⁴ is OR⁷, R¹, R² andR³ are OR⁶, and R⁵ is H.

In an embodiment of Formulas I or III-a to III-e, R⁵ is OR⁷, and R¹-R⁴are OR⁶.

In some embodiments of Formulas I or III-a to III-e, R⁶ is acetyl. Insome embodiments, R⁷ is triflate. And in certain embodiments, R⁶ isacetyl and R⁷ is triflate.

Exemplary Rhamnose analogs according to Formulas III-a to III-e include:

II. Cellobiose analogs

In some embodiments of Formula I, the compound is a cellobiose analog.In such embodiments, R¹ is X, leading to compounds according to FormulaIV:

With respect to Formula IV, R²-R¹¹ are as previously defined for FormulaI, and condition (a) applies. In some embodiments, the cellobiose analogis a D-cellobiose analog.

i) Radiolabeled Cellobiose

In some embodiments of Formula IV, the cellobiose analog is aradiolabeled cellobiose analog, such as an ¹⁸F radiolabeled cellobioseor deoxycellobiose analog. In some embodiments, each of R²-R⁵ and R⁸-R¹¹independently is ¹⁸F or OH, where at least one of R²-R⁵ and R⁸-R¹¹ is¹⁸F. In some embodiments, one of R²-R⁵ and R⁸-R¹¹ is ¹⁸F and the rest ofR²-R⁵ and R⁸-R¹¹ are OH.

In some embodiments, the ¹⁸F radiolabeled cellobiose or deoxycellobioseanalog may have a Formula V

With respect to Formula IV, R²-R⁵ and R⁸-R¹¹ are ¹⁸F or OH, where atleast one, and optionally exactly one of R²-R⁵ and R⁸-R¹¹ is ¹⁸F.

In an embodiment of Formulas IV and V, R² is ¹⁸F and R³-R⁵ and R⁸-R¹¹are OH.

In an embodiment of Formulas IV and V, R³ is ¹⁸F and R², R⁴, R⁵ andR⁸-R¹¹ are OH.

In an embodiment of Formulas IV and V, R⁴ is ¹⁸F and R², R³, R⁵ andR⁸-R¹¹ are OH.

In an embodiment of Formulas IV and V, R⁵ is ¹⁸F and R²-R⁴ and R⁸-R¹¹are OH.

In an embodiment of Formulas IV and V, R⁸ is ¹⁸F and R²-R⁵ and R⁹-R¹¹are OH.

In an embodiment of Formulas IV and V, R⁹ is ¹⁸F and R²-R⁵ and R⁸, R¹⁰and R¹¹ are OH.

In an embodiment of Formulas IV and V, R¹⁹ is ¹⁸F and R²-R⁵ and R⁸, R⁹and R¹¹ are OH.

In an embodiment of Formulas IV and V, R¹¹ is ¹⁸F and R²-R⁵ and R⁸-R¹⁰are OH.

In particular embodiments of Formulas IV and V, R⁹ is ¹⁸F and R²-R⁵ andR⁸, R¹⁰ and R¹¹ are OH, and in another particular embodiment of FormulasIV and V, R² is ¹⁸F and R³-R⁵ and R⁸-R¹¹ are OH.

Exemplary compounds according to Formulas IV and V include:

ii. Radiolabeled cellobiose precursor

In other embodiments of Formula IV, the compound is a precursor compoundto a radiolabeled cellobiose analog. In such embodiments, each of R²-R⁵and R⁸-R¹¹ independently is OR⁶ or OR⁷, where at least one of R²-R⁵ andR⁸-R¹¹ is OR⁷. In some embodiments, one of R²-R⁵ and R⁸-R¹¹ is OR⁷ andthe rest of R²-R⁵ and R⁸-R¹¹ are OR⁶.

The cellobiose precursor analog may have a structure according to anyone of Formulas VI-a to VI-h:

In an embodiment of Formulas IV and VI-a to VI-h, R² is OR⁷ and R³-R⁵and R⁸-R¹¹ are OR⁶.

In an embodiment of Formulas IV and VI-a to VI-h, R³ is OR⁷ and R², R⁴,R⁵ and R⁸-R¹¹ are OR⁶.

In an embodiment of Formulas IV and VI-a to VI-h, R⁴ is OR⁷ and R², R³,R⁵ and R⁸-R¹¹ are OR⁶.

In an embodiment, of Formulas IV and VI-a to VI-h R⁵ is OR⁷ and R²-R⁴and R⁸-R¹¹ are OR⁶.

In an embodiment of Formulas IV and VI-a to VI-h, R⁸ is OR⁷ and R²-R⁵and R⁹-R¹¹ are OR⁶.

In an embodiment of Formulas IV and VI-a to VI-h, R⁹ is OR⁷ and R²-R⁵and R⁸, R¹⁰ and R¹¹ are OR⁶.

In an embodiment, of Formulas IV and VI-a to VI-h R¹⁰ is OR⁷ and R²-R⁵and R⁸, R⁹ and R¹¹ are OR⁶.

In an embodiment of Formulas IV and VI-a to VI-h, R^(H) is OR⁷ and R²-R⁵and R⁸-R¹⁰ are OR⁶.

In particular embodiments of Formulas IV and VI-a to VI-h, R⁹ is OR⁷ andR²-R⁵ and R⁸, R¹⁰ and R¹¹ are OR⁶, and in another particular embodiment,R² is OR⁷ and R³-R⁵ and R⁸-R¹¹ are OR⁶.

In some embodiments of Formulas IV and VI-a to VI-h, R⁶ is acetyl. Insome embodiments, R⁷ is triflate. And in certain embodiments, R⁶ isacetyl and R⁷ is triflate.

Exemplary compounds according to Formulas IV and VI-a to VI-h include:

IV. Compositions

Also provided are compositions that include one or more of theradiolabeled compounds herein, such as an [¹⁸F]-cellobiose or[¹⁸F]-rhamnose compound. In some examples such a composition includes apharmaceutically acceptable carrier, such as water or saline. Thus, insome examples the composition is a liquid composition, for examplesuitable for injection into a subject. In some examples the compositionis frozen or freeze-dried. In some examples, the composition is presentin a container, such as a glass or plastic vial.

V. Methods of Detecting Fungi

Provided here are in vivo and ex vivo/in vitro methods of using one ormore of the radiolabeled compounds provided herein, such as[¹⁸F]-cellobiose or [¹⁸F]-rhamnose, to detect a fungus. The methods candetect any fungus of interest, such as an Aspergillus, Candida,Cryptococcus, or Mucormycetes. In some examples, the method detectsAspergillus, such as A. fumigatus, A. flavus, A. terreus, or A. niger.In some examples, the method detects Candida, such as C. albicans. Insome examples, the method detects Cryptococcus, such as C. neoformans orC. gattii. In some examples, the method detects Mucormycetes, such as aRhizopus, Mucor, Rhizomucor, Syncephalastrum, Cunninghamellabertholletiae, Apophysomyces, or Lichtheimia. The method can includecontacting the fungus in vivo with one or more compounds or compositionsprovided herein, thereby detecting the fungus.

In some examples, the method is an in vivo method of detecting a fungalinfection in a subject (such as any fungus provided above, or listed inTable 1 below; thus in some examples, the subject is one having adisease listed in Table 1). In some examples the subject is a mammal orbird or fish, such a human or veterinary subject. In some examples, thesubject is immunocompromised, such as a cancer patient (e.g., oneundergoing chemo and/or radiation therapy), a subject who has received atransplant (e.g., transplant of at least one of a stem cell or solidorgan such as a lung, heart, liver, kidney, pancreas, or intestine), asubject having a primary immunodeficiency (examples of primaryimmunodeficiency diseases include those listed in Al-Herz et al.(Frontiers in Immunology, volume 5, article 162, April 22, 2014, hereinincorporated by reference in its entirety), e.g., T-B+SCID, T-B- SCID,WHIM syndrome, IL-7 receptor severe combined immune deficiency (SCID),Adenosine deaminase deficiency (ADA) SCID, Purine nucleosidephosphorylase (PNP) deficiency, Wiskott-Aldrich syndrome (WAS), Chronicgranulomatous disease (CGD), Leukocyte adhesion deficiency (LAD),Duchenne muscular dystrophy, Glycogen storage disease type IA, RetinalDystrophy, and X-linked immunodeficiency with magnesium defect,Epstein-Barr virus infection, and neoplasia (XMEN)) or a subject withHIV. In such examples, the contacting includes administering one or morecompounds or compositions provided herein (such as 1, 2, 3, 4 or 5compositions) to a subject, and the method further includes subsequentlyperforming diagnostic imaging (such as nuclear imaging) of the subject,thereby detecting the fungal infection in the subject. For example, thediagnostic imaging can be performed at least 15 minutes, at least 20minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes,or at least 120 minutes, such as 15 to 30, 15 to 60, 30 to 60, 30 to120, or 60 to 120 minutes after administering the one or more compoundsto the subject. In some examples, administering includes injection intothe subject, such as IV administration. In some examples, depending onthe size and weight of the subject, a least 1 millicurie, at least 2millicuries, at least 3 millicuries, at least 4 millicuries, at least 5millicuries, at least 10 millicuries, such as1-3, 1-5, 1-10, 1-20, 5-20or 5-10 millicuries of the one or more compounds is administered to thesubject.

In some examples, diagnostic imaging of the subject includes nuclearimaging of the brain, lungs, heart, sinuses, and/or abdomen of thesubject. In some examples, positron emission tomography (PET) nuclearimaging technology is used. PET enables visualization of metabolicprocesses in vivo. PET imaging detects pairs of gamma rays emittedindirectly by a positron-emitting radionuclide (such as ¹⁸F in[¹⁸F]-cellobiose or [¹⁸F]-rhamnose). PET systems have sensitive detectorpanels to capture gamma ray emissions from inside the body and usesoftware to plot and triangulate the source of the emissions, creating3-D computed tomography images of the tracer concentrations within thebody.

The in vivo methods can he used to detect a fungal infection in asubject. such as in the blood, kidney, heart, esophagus, lungs, sinuses,gastrointestinal tract, and/or central nervous system (e.g., brain,spinal cord). Detection of the administered radiolabeled compound(s)provided herein, such as [¹⁸F]-cellobiose or [¹⁸F]-rhamnose, indicatesthe presence (and location) of a fungal infection. In some examples,such methods are used to monitor treatment of a fungal infection. Thus,in some examples, the subject is one who has previously been treatedwith one or more anti-fungal compositions.

Also provided are ex vivo or in vitro methods of detecting a fungus, forexample by incubating or contacting the one or more radiolabeledcompounds with a sample containing the fungus, for example a biologicalsample obtained from a subject, thereby detecting the fungal infection.The method can further include detecting the uptake of the radiolabeledcompound(s) provided herein, such as [¹⁸F]-cellobiose or [¹⁸F]-rhamnose,in the sample, for example by using a beta counter, radioTLC orautoradiography.

VI. Methods of Treatment

Also provided are methods of treating a subject diagnosed with fungalinfection using the methods provided herein. In some examples, treatmentincludes administering to the subject a therapeutically effective amountof one or more anti-fungal compounds. Any conventional methods ofadministration can be used, such as injection, inhalation, and oraladministration. Exemplary anti-fungal compounds that can be administeredinclude therapeutically effective amounts of one or more ofitraconazole, a corticosteroid, voriconazole, amphotericin B,posaconazole, isavuconazole, caspofungin, micafungin, clotrimazole,miconazole, nystatin, fluconazole, anidulafungin and flucytosine.

Exemplary diseases and treatments are provided in Table 1.

TABLE 1 Fungal diseases affecting patients with weakened immune systemFungus Disease manifestations Method of treatment Aspergillosis Mostcommon is Allergic aspergillosis: Aspergillus fumigatus; Itraconazole+/− corticosteroids can be very aggressive Invasive aspergillosis:especially in Voriconazole + Other options: immunocompromised lipidamphotericin B patients with high formulations, posaconazole, mortalityand isavuconazole, itraconazole, morbidity. caspofungin, and micafunginManifestations include lung disease (invasive pulmonary aspergillosis)and hematogenous spread with potential CNS involvement (cerebralaspergillosis). Both are associated with very high mortalityMucormycosis Rhizopus, Mucor or Amphotericin B, posaconazole, Rhizomucorspecies. or isavuconazole Most commonly affects the sinuses or the lungsafter inhaling fungal spores from the air, or the skin after the fungusenters the skin through a cut, burn, or other type of skin injury.Candidiasis Most common is Mucosal → clotrimazole, Candida albicans.miconazole, or nystatin. For The most serious is severe infections →fluconazole invasive candidiasis Invasive: echinocandin whichoccurs when(caspofungin, micafungin, or Candida species enter anidulafungin) givenIV. the bloodstream or Fluconazole, amphotericin B affect internalorgans may also be appropriate in like the kidney, certain situations.heart, or brain. Cryptococcosis Most common is Mild-to-moderatepulmonary Cryptococcus infections → fluconazole. neoformans. BrainSevere lung infections or CNS infections due to infections →amphotericin B in Cryptococcus are combination with flucytosine, calledcryptococcal followed by long course of meningitis. Most fluconazolecases occur in immunocompromised patients, particularly those who haveadvanced HIV/AIDS, but can also occur in seemingly immunocompetentsubjects.

VII. EXAMPLES Example 1 Synthesis of 2-deoxy-2-fluororhamnose

(3R,4R,5S,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate

Compound 2 was prepared according to the method described by Toyokuni etal., Mol Imaging Biol. 2004, 6(5):324-330. Briefly, L-Rhamnose (10 g,54.9 mmol) was carefully added in portions (3 portions in 15 minutes) toa stirring solution of iodine (0.125 g, 0.49 mmol) in acetic anhydride(60 mL) under a cool water bath (10-15° C.). The resulting mixture wasallowed to warm up to room temperature and stirred for 2 hours. Themixture was then poured on a mixture of crushed ice and saturatedaqueous Na₂S₂O₃ (250 mL, 1:1 mixture) with vigorous stirring. To theresulting light yellow mixture under ice-water bath, NaHCO₃ was addedportion wise until no more CO₂ was released. The crude product wasextracted with CH₂Cl₂ (150 mL×3). The organic layer was combined, washedwith saturated NaHCO3 solution and water (400 mL each), and dried overanhydrous Na₂SO₄. Crude product 2 was obtained by removing the volatilesunder reduced pressure (19.33 g, 95.4% yield, α:β anomer ratio=3:1).

¹H NMR (400 MHz, Chloroform-d) δ 6.02 (d, J=1.9 Hz, 1H), 5.83 (s,0.34H), 5.48 (s, 0.34H), 5.36 -5.28 (m, 1H), 5.26 (dd, J=3.5, 2.0 Hz,1H), 5.18 -5.05 (m, 1.7H), 4.00-3.90 (m, 1H), 3.72-3.62 (m, 0.36H), 2.23(s, 4H), 2.22, (s, 1H), 2.20 (s, 3H), 2.15 (s, 3H), 2.11 (s, 1H), 2.07(s, 4H), 2.01 (s, 4H), 1.30 (d, J=6.2 Hz, 1H), 1.25 (d, J=6.2 Hz, 3H).MS (ESI) calculated mass for the parent C₁₄H₂₀O₉ 332.11 [M], found355.00 [M+Na].

(5S,6S,7R,7aR)-2-ethoxy-2,5-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyldiacetate

To a solution of Compound 2 (19.33 g, 58.2 mmol) in glacial acetic acid(20 mL) and acetic anhydride (1.6 mL) was added HBr in acetic acid (30%,20 mL) dropwise under ice water bath and vigorous stirring. Theresulting mixture was stirred under room temperature overnight andslowly quenched with pre-cooled saturated NaHCO₃ solution (500 mL). Thebrominated intermediate was extracted with CHCl₃ (200 mL×2). The organiclayer was combined, dried over anhydrous Na₂SO₄. The bromideintermediate was obtained by removing the volatiles under reducedpressure as a yellow oil (17.8 g).

The oily bromide intermediate was dissolved in a mixture of anhydrousacetonitrile (8 mL), and 2,4,6-collidine (11 mL), ethanol (200 proof, 13mL) was added. The resulting mixture was stirred under room temperatureovernight, diluted with CH₂Cl₂ (300 mL) and washed water (300 mL×2) andbrine (200 mL). The organic layer was dried over anhydrous Na₂SO₄. Crudeproduct was obtained by removing the volatiles under reduced pressure.Product 3 was purified by flash column chromatography with hexane/ethylacetate 4/1 to 2/1 gradient (7.8 g, 42.2% yield for 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 5.41 (d, J=2.4 Hz, 1H), 5.16-5.02 (m,2H), 4.59 (dd, J=3.8, 2.4 Hz, 1H), 3.65-3.47 (m, 3H), 2.12 (s, 3H), 2.07(s, 3H), 1.75 (s, 3H), 1.30-1.14 (m, 6H). MS (ESI) calculated mass forthe parent Cl₄H₂₂O₈ 318.13 [M], found 341.00 [M+Na].

(3R,4S,5S,6S)-3-hydroxy-6-methyltetrahydro-2H-pyran-2,4,5-triyltriacetate

Hydrochloric acid (1N, 10 mL) was added to a solution of the orthoester3 (7 g, 22.0 mmol) and acetone (15 mL). The mixture was stirred underroom tempertaure for 10 minutes and volatiles were removed under reducedpressure. The resulting crude product was dissolved in CH₂Cl₂ (150 mL)and washed with water (150 mL×2). The organic layer was dried overanhydrous Na₂SO₄. Crude product was obtained by removing the volatilesunder reduced pressure. Product 4 was purified by flash columnchromatography with hexane/ethyl acetate 4/1 to 1/1 gradient (3.15 g,49.3% yield).

¹H NMR (400 MHz, Chloroform-d) δ 5.76 (s, 1H), 5.15 (t, J=9.8 Hz, 1H),4.99 (dd, J=9.9, 3.0 Hz, 1H), 4.22-4.15 (m, 1H), 3.65 (dq, J=9.3, 6.2Hz, 1H), 2.5-2.25 (br s, 1H), 2.17 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H),1.27 (d, J=6.2 Hz, 3H). MS (ESI) calculated mass for the parent C₁₂H₁₈O₈290.10 [M], found 313.00 [M+Na].

(3S,4S,5S,6S)-3-hydroxy-6-methyltetrahydro-2H-pyran-2,4,5-triyltriacetate

The triacetate 4 (1.0 g, 3.19 mmol) was dissolved in anhydrous CH₂Cl₂(20 mL) and anhydrous pyridine (3.5 mL) and cooled with ice-salt bath.Trifluoromethanesulfonic anhydride (4.5 g, 15.97 mmol) in CH₂Cl₂ (10 mL)was added dropwise. The mixture was stirred under room temperature for20 minutes, sequentially washed with HCl (0.3 M, 30 mL), saturatedNaHCO₃ (30 mL) and brine (30 mL). The organic layer was dried overanhydrous Na₂SO₄. The crude triflate was obtained by removing thevolatiles under reduced pressure.

The crude triflate (1.35 g) was stirred with acetonitrile (30 mL) andtetrabutylammonium nitrate (4.59 g, 16.0 mmol) under room temperaturefor 1 hour. Crude product was obtained by removing the volatiles underreduced pressure. Product 5 was purified by flash column chromatographywith hexane/ethyl acetate 3/1 to 1/1 gradient (0.65 g, 65% yield for 2steps). ¹H NMR (400 MHz, Chloroform-d) δ 5.58 (d, J=8.3 Hz, 1H), 5.07(t, J=9.5 Hz, 1H), 4.77 (t, J=9.6 Hz, 1H), 3.76-3.60 (m, 2H), 3.05 (s,1H), 2.15 (s, 3H), 2.07 (s, 5H), 2.04 (s, 4H), 1.20 (d, J=6.2 Hz, 3H).MS (ESI) calculated mass for the parent C₁₂H₁₈O₈ 290.10 [M], found313.00 [M+Na].

(3S,4R,5S,6S)-6-methyl-3-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,4,5-triyltriacetate

The triacetate 5 (0.52 g, 1.79 mmol) was dissolved in anhydrous CH₂Cl₂(20 mL) and anhydrous pyridine (1.2 mL) and cooled with ice-salt bath.Trifluoromethanesulfonic anhydride (1.52 g, 5.37 mmol) in CH₂Cl₂ (10 mL)was added dropwise. The mixture was stirred under room temperature for20 minutes, sequentially washed with HCl (0.3 M, 30 mL), saturatedNaHCO₃ (30 mL) and brine (30 mL). The organic layer was dried overanhydrous Na₂SO₄. Crude product was obtained by removing the volatilesunder reduced pressure. Flash column chromatography was used to purifythe product 6 with hexane / ethyl acetate 3/1 to 1/1 gradient (0.75 g,quant. yield).

¹H NMR (400 MHz, Chloroform-d) δ 5.80 (d, J=8.3 Hz, 1H), 5.38 (t, J=9.6Hz, 1H), 4.89-4.76 (m, 2H), 3.78 (dq, J=9.7, 6.1 Hz, 1H), 2.16 (s, 3H),2.07 (s, 3H), 2.05 (s, 3H), 1.25 (d, J=6.2 Hz, 3H). ¹³C NMR (101 MHz,CDCl₃) δ 169.53, 169.51, 168.44, 118.21 (q, J=319.0 Hz), 90.21, 80.92,77.35, 77.23, 77.03, 76.71, 73.02, 71.27, 71.24, 20.49, 20.37, 20.26,17.02. MS (ESI) calculated mass for the parent C₁₃H₁₇F₃O₁₀S 422.05 [M],found 362.90 [M-OAc].

(3R,4R,5S,6S)-3-fluoro-6-methyltetrahydro-2H-pyran-2,4,5-trioltriacetate

To a solution of triflate 6 (50 mg, 0.118 mmol) in anhydrousacetonitrile (2 mL) was added TBAF in THF (1.0 M, 0.177 mL, 0.177 mmol).The solution was stirred under 65° C. overnight. The volatiles wereremoved under reduced pressure. Flash column chromatography was used topurify the product 7 with hexane/ethyl acetate 5/1 to 2/1 gradient (3.5mg, 10% yield, α: β anomer ratio=1:1). The product characterization wasidentical with the literature.

¹H NMR (400 MHz, Chloroform-d) δ 6.02 (s, 1H), 5.78 (d, J=60 Hz, 1H),5.35-5.30 (m, 1H), 3.30-5.25 (m, 1H), 5.18-5.10 (m, 2H), 5.08-4.95 (m,1H), 4.88 (dd, J=120, 4.0 Hz, 1H), 4.00-3.80 (m, 1H), 3.73-3.65 (m, 1H),2.20 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H),2.02 (s, 3H), 1.30 (d, J=6.0 Hz, 3H), 1.25 (d, J=6.0 Hz, 3H). MS (ESI)calculated mass for the parent C₁₂H₁₇FO₇ 292.10 [M], found 273.10 [M-F].

(3R,4R,5R,6S)-3-fluoro-6-methyltetrahydro-2H-pyran-2,4,5-triol(2-deoxy-2-fluororhamnose)

Triacetate 7 (3.5 mg, 0.012 mmol) was dissolved in TFA (1.0 mL) andstirred under 50° C. for 1 hour. The volatiles were removed underreduced pressure to yield 2-deoxy-2-fluororhamnose as a yellow oil (1.2mg). ¹⁹F NMR chromatogram was compared with literature which foundidentical result. (Liu et al., Chem. Eur. J. 2016, 22, 12557-12565.)

Example 2 Radiosynthesis of 2-deoxy-2-[¹⁸F]fluororhamnose

Radiosynthesis of 2-deoxy-2-[¹⁸F] fluororhamnose was performed on a GETracerlab FX-N2 synthesizer. The synthesis comprised 9 reagent vials onthe GE synthesizer. Vials 1-5 were used for the elution, drying of F-18,and fluorination reaction. Vials 13-14 were used for the formulation ofpurified intermediate, and vials 9-10 for the hydrolysis and formulationof final product [¹⁸F]. Specifically, Vial 1 was added withtetrabutylammonium bicarbonate solution (150 μL, 0.075M), 50 μL waterand MeOH (1 mL); Vial 2 was added with acetonitrile (ACN) (1 mL); Vial 3was added with the tosylate precursor 2 (5 mg) in ACN (0.6 mL); Vial 4was added with water (0.5 mL); Vial 4 was added with 1 mL water; Vial 5was added with HPLC solvent (2.0 mL, 40% ACN in 0.1% trifluoroaceticacid (TFA)); Vial 9 was added with HCl (1N, 700 μL); Vial 10 was addedwith NaOH (1N, 500 μL); Vial 13 was added with EtOH (1.2 mL); Vial 14was added with water (6 mL); HPLC dilution flask was added with water(30 mL). Vial 11 inlet port was connected with valve 15 (V15) right portfor transferring intermediate to reaction vial 2 (RV2).

Typically, 7.4 GBq (200 mCi) [¹⁸F]fluoride in 2.5 mL of water was passedthrough a PS-HCO₃ cartridge, which was rinsed with 1 mL of acetonitrile.[¹⁸F]fluoride was eluted from the cartridge into reactor 1 (R1) with theeluent in Vial 1, and dried under N₂/vacuum at 75° C. for 4 minutes. R1was cooled to 50° C., acetonitrile in Vial 2 was added and the activitywas azeotropically dried at 55° C. for 3 minutes and at 95° C. for 3minutes under N₂/vacuum. The activity was further dried using a vacuumfor 3 minutes. The [¹⁸F]fluoride drying cycle took about 20 minutes.

The tosylate precursor solution in Vial 3 was added to the driedactivity. The resulting solution was stirred at 70° C. for 20 minutes,then cooled to 45° C. The reaction mixture was diluted with 1.0 mL ofwater (Vial 4), and transferred in Tube 2. R1 was rinsed with HPLCmobile phase (Vial 5) and the solution was also transferred to Tube 2.The solution in Tube 2 was thoroughly mixed by bubbling N₂ for 10seconds and injected into the HPLC for purification. HPLC condition:Phenomenex Luna (2) C18 column, 250×10 mm, 5 μm. Mobile phase: 40% ACNin 0.1% TFA. Flow rate: 4 mL/min. The labeled intermediate was eluted atabout 12-14 minutes. The intermediate was collected in the dilutionflask containing 30 mL water, and passed through an Oasis HLB pluscartridge (pre-conditioned with 5 mL of ethanol, 10 mL of air, and 10 mLof water). The trapped intermediate was rinsed with 6 mL water (Vial14), and eluted with 1.2 mL of absolute ethanol (Vial 13) to R2 throughvalue 35 (V35).

The eluted intermediate was heated to 60° C. under N₂ flow and vacuumfor 3 minutes to remove ethanol. NaOH in Vial 10 was added to the driedresidue. The resulting solution was heated at 45° C. for 10 minutes. HClsolution in Vial 9 was added, the content was transferred through V16and an in-line sterile filter to the product vial. The product wasanalyzed by HPLC. HPLC conditions: Waters BEH Amide column (150*4.6 mm),3.5 μm. Using 90% −50% D in 8 minutes. C: 95% water +5% ACN with 0.1%NH₄OH; D: 95% ACN +5% water with 0.1% NH₄OH. The product peak eluted atabout 4 minutes. Generally, 2-deoxy-2-[¹⁸F] fluororhamnose 9 wassynthesized in 7-12% radiochemical yield (uncorrected, n>5),radiochemical purity >95%. The synthesis time was about 90 minutes.

A manual method also was used to produce the radiolabeled product.Different bases were tried and produced various overall yields.

Precursor Temp. Time RCY (mg) Base (° C.) (min) (%) 3 K₂CO₃ (2 mg) 10010 n/a 3 K₂CO₃ (2 mg) 60 20 n/a 3 TBAB (100 μL, 60 20 10-34 0.075M) 3TBAB (100 μL, 45 20 20-50 0.075M) 3 TBAB (100 μL, rt 20 5% 0.075M) 5“Rxn on Sep-pak” n/a method

Example 3 Synthesis of 6-F-Rhamnose

(3S,5S,6R)-6-((trityloxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

Triphenylmethyl chloride (3.4 g, 12.2 mmol) was added to L-Mannose 10(2.00 g, 11.1 mmol) in anhydrous pyridine (10 mL). The mixture wasstirred at room temperature for 15 hours. 6 mL of Ac₂O was addedafterwards and the solution was stirred for another 15 hours. Themixture was poured into ice-cold water and extracted with EtOAc (3×100mL). The combined organic layer was washed with brine and dried overNa₂SO₄. After evaporation of solvents, the residue was purified bysilica gel flash chromatography to afford the product 11 as a whitesolid (5.84 g, 89%).

¹H NMR (400 MHz, CDCl₃): δ 7.46-7.22 (m, 15H), 6.10 (s, 0.7H), 5.85 (s,0.3H), 5.52 (m, 1H), 5.43-5.52 (m, 2H), 3.91 (m, 0.7H), 3.64 (m, 0.3H),3.34 (m,1H), 3.18 (0.3H), 3.07 (m, 0.7H), 2.24 (s, 2.1H), 2.23 (s,0.9H), 2.17 (s, 2.1H), 2.14 (s, 0.9H), 2.00 (s, 2.1H), 1.98 (s, 0.9H),1.76 (s, 0.9H), 1.75 (s, 2.1H).

(3S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

33% HBr in HOAc (1.6 mL) was added to the solution of compound 11 (4.60g, 7.80 mmol) in glacial acetic acid (16 mL) at 10° C. The mixture wasstirred for 10 minutes. The formed triphenylmethyl bromide wasimmediately removed by filtration. The filtrate was diluted with coldwater and extracted with EtOAc (3×100 mL). The combined organic layerwas washed with water, brine and dried over Na₂SO₄. After evaporation ofsolvents, the residue was purified by silica gel flash chromatography toafford the product 12 as a white solid (2.23 g, 82%).

¹H NMR (400 MHz, CDCl₃): δ 6.09 (d, 0.67H, J=1.6 Hz), 5.87 (d, 0.33 H,J=1.2 Hz), 5.49 (dd, 0.33 H, J=1.2 and 11.5 Hz), 5.40 (dd, 0.67 H, J=3.3and 10.0 Hz), 5.33 (m, 0.67H), 5.27 (m, 1H), 5.17 (dd, 0.33 H, J=3.3 and10.5 Hz), 3.85 (m, 0.67H), 3.73 (m, 1H), 3.66-3.58 (m, 1.33H), 2.21 (s,0.99H), 2.17 (s, 2.01H), 2.16 (s, 2.01H), 2.10 (s, 0.99H), 2.08 (s,2.01H), 2.04 (s, 0.99H), 2.02 (s, 2.01H), 2.01 (s, 0.99H).

3S,5S,6R)-6-((((trifluoromethyl)sulfonyl)oxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

Trifluoromethanesulfonic anhydride (0.37 mL, 2.2 mmol) was added to amixture of compound 12 (696 mg, 2.0 mmol) and pyridine (0.25 mL) indichloromethane (20 mL) at -10° C. After stirring for 2 hours, water (50mL) was added. The organic layer was separated and the aqueous layer wasextracted with dichloromethane (3×50 mL). The organic layers werecombined, washed with 10% H₂SO₄, sat. NaHCO₃, brine and dried overMgSO₄. After evaporation of solvents, the residue was purified by silicagel flash chromatography to afford the product 13 as a white solid (826mg, 86%).

¹H NMR (400 MHz, CDCl₃): δ 6.12 (d, 0.62H, J=2.0 Hz), 5.89 (d, 0.38 H,J=1.2 Hz), 5.49 (dd, 0.38 H, J=1.2 and 3.1 Hz), 5.39 (dd, 0.62 H, J=3.1and 10.2 Hz), 5.33 (m, 0.62H), 5.30 (m, 0.38H), 5.26 (dd, 0.62H, J=1.2and 11.5 Hz), 5.16 (dd, 0.38 H, J=3.1 and 9.8 Hz), 4.58-4.54 (m, 2H),4.14 (m, 0.62H), 3.92 (m, 0.38), 2.22 (s, 1.14H), 2.19 (s, 1.86Hx2),2.12 (s, 1.14H), 2.10 (s, 1.86H), 2.05 (s, 1.14H), 2.03 (s, 1.86H), 2.02(s, 1.14H). ¹⁹F NMR (376 MHz, CDCl₃): δ-74.3-74.4.

(3S,4R,5R,6R)-6-(fluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

DAST (0.30 mL, 2.27 mmol) was slowly added to a solution of 13 (104 mg,0.30 mmol) in anhydrous CH₂Cl₂ (5 mL) at −40° C. The reaction wasstirred at room temperature for 24 hours. After cooled down to −20° C.,MeOH (1 mL) was added and the solvent was removed under reducedpressure. The residue was diluted with CH₂Cl₂ (75 mL), washed with waterand dried over MgSO₄. After evaporation of solvents, the residue waspurified by silica gel flash chromatography to afford the product 14 asa colorless oil (30 mg, 28%). ¹⁹F NMR (376 MHz, CDCl₃): δ-231.9, -232.4.

(3S,4R,5R,6R)-6-(fluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol

NaOMe (13.5 mg, 0.25 mmol) was added to a suspension of 14 (22 mg, 0.063mmol) in dry MeOH (3 mL). The mixture was stirred at room temperaturefor 15 hours. Then the reaction mixture was neutralized with Dowex (H+)resin, filtrated, concentrated and purified by silica gel flash columnchromatography to afford 15 as a white solid (10 mg, 87%).

¹H NMR (400 MHz, D₂O/CD₃OD): δ 5.18 (d, 0.6H, J=2.0 Hz), 4.92 (d, 0.4 H,J=1.2 Hz), 4.78-4.57 (m, 2H), 3.93 (m, 1H), 3.87 (m, 1H), 3.77 (m, 1H),3.68 (m, 1H).

Example 4 Radiosynthesis of 6-[¹⁸F]fluororhamnose

Radiosynthesis of 6-[¹⁸F] fluororhamnose was performed on a GE TracerlabFX-N2 synthesizer. The synthesis comprised 9 reagent vials on the GEsynthesizer. Vials 1-5 were used for the elution, drying of F-18, andfluorination reaction. Vials 13-14 were used for the formulation ofpurified intermediate, and vials 9-10 for the hydrolysis and formulationof final product [¹⁸F]16. Specifically, Vial 1 was added withtetrabutylammonium bicarbonate solution (150 μL, 0.075M), 50 μL waterand MeOH (1 mL); Vial 2 was added with ACN (1 mL); Vial 3 was added withthe tosylate precursor 2 (5 mg) in ACN (0.6 mL); Vial 4 was added withwater (0.5 mL); Vial 4 was added with 1 mL water; Vial 5 was added withHPLC solvent (2.0 mL, 40% ACN in 0.1% TFA); Vial 9 was added with HCl(1N, 700 μL); Vial 10 was added with NaOH (1N, 500 μL); Vial 13 wasadded with EtOH (1.0 mL); Vial 14 was added with water (6 mL); HPLCdilution flask was added with water (30 mL). Vial 11 inlet port wasconnected with V15 right port for transferring intermediate to RV2.

Typically, 7.4 GBq (200 mCi) [¹⁸F] fluoride in 2.5 mL of water waspassed through a PS-HCO₃ cartridge, which was rinsed with 1 mL ofacetonitrile. [¹⁸F]fluoride was eluted from the cartridge into reactor 1(R1) with the eluent in Vial 1, and dried under N₂/vacuum at 75° C. for4 minutes. R1 was cooled to 50° C., acetonitrile in Vial 2 was added andthe activity was azeotropically dried at 55° C. for 3 minutes and at 95°C. for 3 minutes under N2/vacuum. The activity was further dried using avacuum for 3 minutes. The [¹⁸F]fluoride drying cycle took about 20minutes.

The tosylate precursor solution in Vial 3 was added to the driedactivity. The resulting solution was stirred at 70° C. for 20 minutes,cooled to 45° C. The reaction mixture was diluted with 1.0 mL of water(Vial 4), and transferred in Tube 2. R1 was rinsed with HPLC mobilephase (Vial 5) and the solution was also transferred to Tube 2. Thesolution in Tube 2 was thoroughly mixed by bubbling N2 for 10 secondsand injected into the HPLC for purification. HPLC condition: PhenomenexLuna (2) C18 column, 250×10 mm, 5 μm. Mobile phase: 40% ACN in 0.1% TFA.Flow rate: 4 mL/min. The labeled intermediate was eluted at about 12-14minutes. The intermediate was collected in the dilution flask containing30 mL water, and passed through an Oasis HLB light cartridge(pre-conditioned with 5 mL of ethanol, 10 mL of air, and 10 mL ofwater). The trapped intermediate was rinsed with 6 mL water (Vial 14),and eluted with 1.0 mL of absolute ethanol (Vial 13) to R2 through V35.

The eluted intermediate was heated to 60° C. under N₂ flow and vacuumfor 3 minutes to remove ethanol. NaOH in Vial 10 was added to the driedresidue. The resulting solution was heated at 45° C. for 10 minutes. HClsolution in Vial 9 was added, the content was transferred through valve16 (V16) and an in-line sterile filter to the product vial. The productwas analyzed by HPLC: Waters BEH Amide column (150*4.6 mm), 3.5 μm.Using 90% −50% D in 8 min. C: 95% water +5% ACN with 0.1% NH₄OH; D: 95%ACN +5% water with 0.1% NH₄OH. The product peak eluted at about 5minutes. Generally, 6-[18F]fluororhamnose was synthesized in 18-25%radiochemical yield (uncorrected, n >3), radiochemical purity >95%. Thesynthesis time was about 90 minutes.

Example 5 Synthesis of 3-deoxy-3-F-Rhamnose

(3R,4R,5S,6S)-4-(benzyloxy)-2-methoxy-6-methyltetrahydro-2H-pyran-3,5-diol

Methyl-rhamnopyranoside 17 (2.85 g, 16.0 mmol), benzyl bromide (2.91 mL,24 mmol), dimethyltin dichloride (351 mg, 1.6 mmol) and Ag₂O (4.07 g,17.6 mmol) were stirred in anhydrous acetonitrile (90 mL) at roomtemperature for 15 hours. After filtered through a celite pad, thefiltrate was evaporated and the residue was purified by silica gel flashchromatography to afford 18 as a colorless oil (3.41 g, 79%, α:β1).

β-isomer: ¹H NMR (400 MHz, CDCl₃) δ 7.30-7.32 (m, 5H), 4.71 (d, 1H,J=1.6 Hz), 4.70 (d, 1H, J=11.3 Hz), 4.57 (d, 1H, J=11.3 Hz), 4.02 (dd, 1H, J=1.6 and 3.1 Hz), 3.67-3.61 (m, 2H), 3.56 (m, 1 H), 3.36 (s, 3H),1.32 (d, J=6.3 Hz, 3H).

α-isomer: ¹H NMR (400 MHz, CDCl₃) δ 7.39-7.32 (m, 5H), 4.75 (d, 1H,J=11.3 Hz), 4.74 (s, 1H), 4.52 (d, 1H, J=11.7 Hz), 3.72-3.68 (m, 2H),3.60 (m, 1 H), 3.42 (t, 1H, J=9.0 Hz), 3.35 (s, 3H), 1.34 (d, J=6.3 Hz,3H).

(3R,4R,5S,6S)-4-(benzyloxy)-5-hydroxy-6-methyltetrahydro-2H-pyran-2,3-diyldiacetate

Compound 18 (3.24 g, 12.1 mmol) was dissolved in anhydrous pyridine (12mL) and Ac₂O (7 mL). The solution was stirred at room temperature for 15hours. Solvents were evaporated and the residue was dissolved in EtOAc(300 mL), washed with saturated NaHCO₃, 1N HCl, H₂O, brine and driedover Na₂SO₄. After evaporation of solvents, the crude product 19 wasused for next step.

H₂SO₄ (0.6 mL) was added dropwise to a solution of 19 (4.25 g, 12.1mmol) in Ac₂O (20 mL) and the solution was stirred at room temperaturefor 5 hours. The reaction mixture was poured into a stirred mixture ofethyl acetate (150 mL) and saturated NaHCO₃ (80 mL). The organic phasewas separated and washed with saturated NaHCO₃, brine and dried overNa₂SO₄. After evaporation of solvents, the residue was purified bysilica gel flash chromatography to afford the product 20 as a colorlessoil (3.37 g, 73%).

¹H NMR (400 MHz, CDCl₃): δ 7.37-7.26 (m, 5H), 6.12 (d, 0.27H, J=2.0 Hz),6.03 (d, 0.73H, J=2.0 Hz), 5.34 (dd, 0.73H, J=2.0 and 3.5 Hz), 5.23 (m,0.27H), 5.16 (m, 0.27H), 5.07 (t, 0.73H, J=9.0 Hz), 4.72-4.43 (m, 2H),3.94-3.79 (m, 2H), 2.16 (s, 2.19H), 2.12 (s, 0.81H), 2.11 (s, 2.19H),2.10 (s, 0.81H), 2.05 (s, 0.81H), 2.04 (s, 2.19H), 1.23 (d, J=6.3 Hz,0.81H), 1.21(d, J=6.3 Hz, 2.19H).

(3R,4R,5R,6S)-4-hydroxy-6-methyltetrahydro-2H-pyran-2,3,5-triyltriacetate

10% Pd/C (1.5 g) was added to 20 (3.15 g, 8.28 mmol) in EtOAc (200 mL).The mixture was stirred at room temperature under H₂ atmosphere for 2hours and filtered through a celite pad.

The filtrate was evaporated and the residue was purified by silica gelflash chromatography to afford 21 as a white solid (2.14 g, 89%).

¹H NMR (400 MHz, CDCl₃): δ 6.10 (d, 0.26H, J=2.0 Hz), 6.06 (d, 0.74H,J=1.6 Hz), 5.25 (dd, 0.26H, J=3.1 and 9.8 Hz), 5.17 (m, 0.26H), 5.09(dd, 0.74H, J=1.8 and 13.7 Hz), 4.90 (t, 0.74H, J=9.8 Hz), 4.10-4.00 (m,1H), 3.97-3.84 (m, 1H), 2.16 (s, 2.22H), 2.12 (s, 0.78H), 2.11 (s,2.22H), 2.10 (s, 0.78H), 2.05 (s, 0.78H), 2.04 (s, 2.22H), 1.23 (d,J=6.3 Hz, 0.78H), 1.21(d, J=6.3 Hz, 2.22H).

(3R,4R,5S,6S)-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,3,5-triyltriacetate

Trifluoromethanesulfonic anhydride (0.33 mL, 1.94 mmol) was added to amixture of compound 21 (508 mg, 1.75 mmol) and pyridine (0.22 mL) indichloromethane (18 mL) at -18° C. After stirring for 0.5 hours, themixture was warmed up to room temperature and stirred for additional 0.5hours. Water (50 mL) was added and the organic layer was separated. Theaqueous layer was extracted with dichloromethane (3×50 mL). The combinedorganic layers were washed with 10% H₂SO₄, saturated NaHCO₃, brine anddried over MgSO₄. After evaporation of solvents, the residue waspurified by silica gel flash chromatography to afford the product 22 asa colorless oil (494 mg, 67%).

¹H NMR (400 MHz, CDCl₃): δ 6.06 (d, 1H, J=2.0 Hz), 5.38 (dd, 1H, J=2.0and 3.5 Hz), 5.28 (t, 1H, J=9.8 Hz), 5.18 (dd, 1H, J=3.7 and 10.0 Hz),3.92 (m, 1H), 2.21 (s, 3H), 2.18 (s, 3H), 2.15 (s, 3H), 1.27 (d, J=6.3Hz, 3H). ¹⁹F NMR (376 MHz, CDCl₃): δ-75.0.

(3R,4S,5R,6S)-4-hydroxy-6-methyltetrahydro-2H-pyran-2,3,5-triyltriacetate

Compound 22 (422 mg, 1.0 mmol) was dissolved in dry CH₃CN (2 mL) andsolid tetrabutylammonium nitrite (1.44 g, 5 mmol) was added. Afterstirring for 1 hour at room temperature, the reaction mixture wasevaporated. The residue was dissolved in CH₂Cl₂, washed with brine anddried over MgSO₄. After evaporation of solvents, the residue waspurified by silica gel flash chromatography to afford the product 23 asa white solid (87 mg, 30%).

¹H NMR (400 MHz, CDCl₃): δ 5.95 (d, 0.84H, J=2.3 Hz), 5.91 (s, 0.16H),5.10 (m, 0.16H), 5.02 (dd, 0.16H, J=1.6 and 3.5 Hz), 5.00 (dd, 0.84H,J=2.3 and 4.3 Hz), 4.89 (dd, 0.84H, J=3.3 and 8.8 Hz), 4.27 (m, 0.84H),4.12-4.06 (m, 1H), 3.74 (m, 0.16H), 2.15 (s, 0.48H), 2.13 (s, 0.84×6H),2.12 (s, 0.84×3H), 2.11 (s, 0.48H), 2.10 (s, 0.48H), 1.33 (d, J=6.7 Hz,0.48H), 1.25(d, J=6.7 Hz, 0.84×3H).

(3R,4S,5S,6S)-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,3,5-triyltriacetate

Compound 23 is treated with trifluoromethanesulfonic anhydride andpyridine to make compound 24 according to the method previouslydescribed with respect to the synthesis of compound 22.

(3S,4R,5S,6S)-4-fluoro-6-methyltetrahydro-2H-pyran-2,3,5-triyltriacetate

Compound 24 is treated with DAST to make compound 25 according to themethod previously described with respect to the synthesis of compound14.

(3S,4R,5S,6S)-4-fluoro-6-methyltetrahydro-2H-pyran-2,3,5-triol

Compound 25 is treated with NaOMe to make compound 26 according to themethod previously described with respect to the synthesis of compound15.

Example 6 Radiosynthesis of 3-deoxy-3-[¹⁸F]-fluororhamnose

3-deoxy-3-[¹⁸F]-fluororhamnose is synthesized from compound 24 using themethod previously described, such as in Examples 2 and 4.

Example 7 Synthesis of(3R,4R,5S,6R)-5-(((2S,3R,4S,5S,6R)-3-[¹⁸F]-fluoro-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4-triol)

(2R,3R,4S,5S,6S)-2-(Acetoxymethyl)-5-(benzyloxy)-6-(((1R,2R,3S,4R)-3,4-diacetoxy-6,8-dioxabicyclo[3.2.1]octan-2-yl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate (29)

A solution of compound 27 in anhydrous ether is added slowly to astirred solution of compound 28 in anhydrous acetonitrile containingsilver trifluoromethanesulfonate (CF₃SO₃Ag), silver carbonate (Ag₂CO₃)and powdered drierite. The resulting mixture is stirred at roomtemperature and filtered through celite. The residue is washed withCH₂Cl₂. The combined filtrates are evaporated and the residue ispurified by silica gel flash chromatography to produce compound 29.

(3R,4S,5R,6R)-6-(Acetoxymethyl)-5-(((2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-(benzyloxy)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2,3,4-triyltriacetate (30)

Compound 29 is treated with Ac₂O and H₂SO₄ to make compound 30, usingthe method previously described in the synthesis of compound 20.

(3R,4S,5R,6R)-6-(Acetoxymethyl)-5-(((2S,3S,4R,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-hydroxytetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2,3,4-triyltriacetate (31)

Compound 30 is treated with 10% Pd/C and H2 to make compound 31, usingthe method previously described in the synthesis of compound 21.

(3R,4S,5R,6R)-6-(Acetoxymethyl)-5-(((2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2,3,4-triyltriacetate (32)

Compound 31 is treated with trifluoromethanesulfonic anhydride andpyridine to make compound 32, using the method previously described inthe synthesis of compound 22.

(3R,4R,5S,6R)-5-(((2S,3R,4S,5S,6R)-3-Fluoro-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4-triol(33)

n-Bu₄NF is added to a solution of compound 32 in anhydrous acetonitrile.The reaction mixture is stirred at room temperature, concentrated andthe residue is purified by flash chromatography on a silica gel columnto give the intermediate product which is treated with NaOMe to producecompound 33, using the method previously described in the synthesis ofcompound 15.

(3R,4R,5S,6R)-5-(((2S,3R,4S,5S,6R)-3[¹⁸F]-fluoro-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4-triol(8-deoxy-8[¹⁸F]-fluorocellobiose)

8-deoxy-8[¹⁸F]-fluorocellobiose is synthesized from compound 32 usingthe methods described herein.

Example 8 Synthesis of(2S,3R,4S,5S,6R)-2-(((2R,3S,4S,5R,6R)-5-[¹⁸F]-fluoro-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol)

(3S,4S,5R,6R)-6-(Acetoxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2,3,4-triyl triacetate(35)

Compound 34 is treated with Ac₂O and pyridine to make compound 35, usingthe method previously described in the synthesis of compound 19.

(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(((2R,3R,4S,5S,6R)-4,5-diacetoxy-2-(acetoxymethyl)-6-iodotetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (36)

Iodine and triethylsilane are sequentially added to a solution ofcompound 35 in dichloromethane. The reaction is stirred at refluxtemperature until TLC analysis indicates that the reaction issubstantially complete. After cooling to room temperature, the mixtureis diluted with dichloromethane and washed with a solution of saturatedsodium hydrogen carbonate containing sodium thiosulfate for reducing theresidual amount of iodine. The aqueous phase is extracted withdichloromethane, and the collected organic phase is dried with sodiumsulfate and concentrated to provide the crude product 36 for next step.

(2S,3R,4S,5R,6R)-2-(((3aS,5R,6R,7S,7aS)-7-Acetoxy-5-(acetoxymethyl)-2-(benzyloxy)-2-methyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6-yl)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (37)

Freshly activated 4 A molecular sieves are added to the residue, and themixture is suspended in anhydrous dichloromethane. 2,4,6-collidine andBnOH are then added, and the mixture is stirred at room temperature. Themixture is filtered through a short pad of silica gel, and the residuefrom the filtered liquor is purified by silica gel flash chromatographyto afford the product 37.

(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(((2R,3R,4R,5S,6S)-4,6-diacetoxy-2-(acetoxymethyl)-5-hydroxytetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (38)

Compound 37 is treated with 10% Pd/C and H2 to make compound 38, usingthe method previously described in the synthesis of compound 21.

(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(((2R,3R,4S,5S,6S)-4,6-diacetoxy-2-(acetoxymethyl)-5-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-trioltriacetate (39)

Compound 38 is treated with trifluoromethanesulfonic anhydride andpyridine to make compound 39, using the method previously described inthe synthesis of compound 22.

(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(((2R,3R,4S,5R,6S)-4,6-diacetoxy-2-(acetoxymethyl)-5-fluorotetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (40)

Compound 39 is treated with n-Bu₄NF to make compound 40, using themethod previously described in the synthesis of compound 33.

(2S,3R,4S,5S,6R)-2-(((2R,3S,4S,5R,6R)-5-Fluoro-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol(41)

Compound 40 is treated with NaOMe to make compound 41 using the methodpreviously described in the synthesis of compound 15.

(2S,3R,4S,5S,6R)-2-(((2R,3S,4S, 5R,6R)-5-[¹⁸F]-fluoro-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol(2-2[¹⁸F]-fluorocellobiose)

2-deoxy-2[¹⁸F]-fluorocellobiose is synthesized from compound 39 usingthe methods described herein.

Example 9 L-Rhamnose as a Marker for Fungal Infection

L-Rhamnose uptake is low in bacteria (gram negative and gram positive)and very low in P. aeruginosa and macrophages (Ordonez et al., J. NuclMed, 58: 144-50, 2017). L-Rhamnose is metabolized in Aspergillus speciesby alpha-rhamnosidases. Fungi such as Aspergillus use anonphosphorylative pathway where L-Rhamnose is first oxidized toL-rhamno-g-lactone by L-rhamnose-1-dehydrogenase (LRA1). Thisintermediate is then converted to L-rhamnonate by L-rhamnono-g-lactonase(LRA2) and subsequently to L-2-keto-3-deoxyrhamnonate by L-rhamnonatedehydratase (LRA3). This is then cleaved into pyruvate andL-lactaldehyde by L-2-keto-3-deoxyrhamnonate (Watanabe et al., Febs J,275:5139-49, 2008; Watanabe et al., J. Biol Chem, 283:20372-82, 2008).

Radiolabeled L-Rhamnose was tested as a fungal-specific ligand. In vitrouptake assays using 3H-L-Rhamnose (in vitro incubation of ³H-L-Rhamnosewith different cultures of Aspergillus, Rhizomucor, Cryptococcus,bacteria and macrophages followed by culture washing and measurement ofretained radioactivity) showed significant retention of the sugar by A.fumigatus (FIG. 1A), while minimal retention was seen with bacteria andmacrophages (FIGS. 1B-1D).

ex vivo experiments were performed, including biodistribution (injectingthe animals with radiolabeled molecules followed by euthanasia,collection of organs and measurement of radioactivity) andautoradiography (sectioning of lung tissues from injected animals tomeasure residual radioactivity). Biodistribution studies showed uptakeof ³H-L-Rhamnose in the lungs of infected animals but not in uninfectedcontrols (FIG. 2). Autoradiography showed accumulation of ³H-L-Rhamnosein the lungs of infected but not uninfected animals (FIG. 1F).

¹⁸F-L-rhamnose PET ligand (¹⁸F-2-deoxy-2-fluoro-L-hamnose;radiosynthesis details provided separately by the CSC) was synthesizedas described in Examples 1 and 2, and evaluated for in vitro uptake byA. fumigatus and E. coli. ¹⁸F-L-rhamnose was specifically internalizedby live A. fumigatus cultures when compared to heat-killed cultures butnot by E. coli. in vivo uptake of ¹⁸F-L-rhamnose was assessed by PET/CTin mouse models of pulmonary aspergillosis (2 days followingpost-pharyngeal inoculation with Aspergillus cultures). Standardizeduptake values (SUVs) of ¹⁸F-L-rhamnose in infected mice were thenmeasured, and compared to animals with sterile lung inflammation (24hours following post-pharyngeal poly (I:C) administration) and healthycontrols. In vivo PET/CT imaging with a 60-minute dynamic ¹⁸F-L-rhamnosePET/CT imaging of a pulmonary aspergillosis model showed a slight higheruptake in lung lesions compared to controls and poly (I:C) treated mice(FIG. 3A). There was however relatively fast washout of the ligand (FIG.3B).

These results demonstrate that A. fumigatus selectively accumulates¹⁸F-L-rhamnose in vitro and in vivo.

Based on these observations, ¹⁸F-L-rhamnose derivatives with the ¹⁸Flocated on other carbon atoms instead of C2, for example, C3 or C6, canbe generated as described herein. These resulting ¹⁸F-L-rhamnosederivatives (see Examples, 3-6) can be used to detect fungi in vivo orex vivo using the methods provided herein. For example, dynamic PET/CTimaging and delayed PET/CT imaging (acquiring 60 minutes of dynamic dataafter injection of the ligand, and static imaging at 120 minutes postinjection) can be performed. Bolus/infusion can be used to prolong thebiological half-life of the compound and increase circulation time whichwould result in higher uptake by the fungi.

Example 10 Cellobiose as a Marker for Fungal Infection

Cellobiose, a disaccharide (two β-glucose molecules with a 1→4glycosidic bond), is metabolized by Aspergillus fumigatus β-glucosidaseand has no known human metabolism. In the presence of β-glucosidase,cellobiose is metabolized into two glucose molecules resulting in uptakein the area of infection as well as release of glucose into thecirculation resulting in brain uptake (cellobiose does not cross theblood brain barrier, BBB). Thus, cellobiose can be radiolabeled and usedas a ligand for fungal detection.

In vitro studies using ³H-cellobiose were performed with measurement ofretained radioactivity after incubation and washing of the culturesusing a beta counter. A. fumigatus had high uptake of ³H-cellobiose(>than 2-deoxyglucose uptake) in culture (FIG. 4A) while mammalian cellsdid not (FIG. 4B). This uptake of ³H-cellobiose in A. fumigatusincreased over time, especially at 120 minutes.

In biodistribution studies using ³H-cellobiose (animals injected with³H-cellobiose followed by euthanasia, collection of organs andmeasurement of radioactivity), increased uptake in A. fumigatus-infectedlungs (nasopharyngeal model) was found, as compared to uninfectedanimals (FIG. 4C). Uptake in the brain was also noted innasopharyngeally infected animals indicating hydrolysis of ³H-cellobioseinto two glucose molecules and secondary uptake of glucose molecules bybrain cells.

Uptake of ³H-cellobiose by the infected animal lungs (nasopharyngealinoculation model) and brains (intravenous inoculation model), but notin control animals or animals with sterile lung inflammation byautoradiography (sectioning of lung tissues from injected animals tomeasure retained radioactivity), was observed (FIGS. 5A and 5B). Thosefindings were supported by GMS staining showing fungal hyphae in similardistributions to autoradiography uptake.

To demonstrate the specificity of ³H-cellobiose, in vitro screening ofvarious bacteria can be performed with radioTLC to confirm the presenceor lack of cellobiose metabolism. RadioTLC can be performed using³H-cellbiose as a reference and the presence of labeled glucose in thecell lysate obtained following incubation with fungi or bacteria canthen be determined. If the pathogen has β-glucosidase, more than onepeak will be detected, indicating metabolism (glucose and downstreammetabolites); if the pathogen does not have β-glucosidase, only one peakrepresenting the parent molecule (cellobiose), is detected. Radio-TLCcan be used to evaluate plasma from control mice to further confirm thelack of mammalian metabolism for cellobiose: if β-glucosidase isexpressed in the mouse, more than one peak will be detected, indicatingmetabolism (glucose and downstream metabolites) but if the enzyme is notpresent, as expected, only radiolabeled cellobiose will be detected.

Radiosynthesis of labeled cellobiose can be achieved with ¹⁸F located onone of the carbon atoms on the cellobiose molecule, for example, on C2in the first or second glucose molecule (FIG. 6; see Examples 7 and 8).The in vitro uptake of both ¹⁸F-cellobiose molecules can be measured asdescribed herein and used for in vivo PET imaging as described herein.Since the resulting molecule is essentially FDG (after breaking the 1→4glycosidic bond by β-glucosidase), this will result in uptake,phosphorylation and entrapment of ¹⁸F-cellobiose metabolites in areaswith fungal infection.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the invention and should not be takenas limiting the scope of the invention. Rather, the scope of theinvention is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

1-44 (canceled)
 45. A compound having a structure according to FormulaII or Formula V

wherein: with respect to Formula II each of R², R³, and R⁴ independentlyis ¹⁸F or OH; R⁵ is ¹⁸F or H; and at least one of R¹-R⁵ is ¹⁸F; and withrespect to Formula V each of R²-R⁵ and R⁸-R¹¹ independently is ¹⁸F orOH; and at least one of R²-R⁵ and R8-R¹¹ is ¹⁸F.
 46. The compound ofclaim 45, wherein the compound has a structure according to Formula II.47. The compound of claim 46, wherein the compound is


48. The compound of claim 45, wherein the compound has a structureaccording to Formula V.
 49. The compound of claim 48, wherein one ofR²-R⁵ and R⁸-R¹¹ is ¹⁸F and the rest of R²-R⁵ and R⁸-R¹¹ are OH.
 50. Thecompound of claim 48, wherein the compound is selected from


51. A compound having a formula selected from

wherein: R⁶ is acetyl, formyl, methoxyacetyl, benzoyl, haloacetyl ortrialkylsilyl; and R⁷ is triflate, mesylate or tosylate.
 52. Thecompound of 51, wherein the compound is selected from


53. A compound having a structure according to Formula IV

wherein: each of R², R³, R⁴, R⁸, R⁹, R¹⁰ and R¹¹ independently is OR⁶ orOR⁷; R⁶ is acetyl, formyl, methoxyacetyl, benzoyl, haloacetyl ortrialkylsilyl; R⁷ is triflate, mesylate or tosylate; and at least one ofR²-R⁵ and R⁸-R¹¹ is OR⁷ and the remainder are OR⁶.
 54. The compound ofclaim 53, wherein: R² is OR⁷ and R³-R⁵ and R⁸-R¹¹ are OR⁶; R³ is OR⁷ andR², R⁴, R⁵ and R⁸-R¹¹ are OR⁶; R⁴ is OR⁷ and R², R³, R⁵ and R⁸-R¹¹ areOR⁶; R⁵ is OR⁷ and R²-R⁴ and R⁸-R¹¹ are OR⁶; R⁸ is OR⁷ and R²-R⁵ andR⁹-R¹¹ are OR⁶; R⁹ is OR⁷ and R²-R⁵ and R⁸, R¹⁰ and R¹¹ are OR⁶; R¹⁰ isOR⁷ and R²-R⁵ and R⁸, R⁹ and R¹¹ are OR⁶; or R¹¹ is OR⁷ and R²-R⁵ andR⁸-R¹⁰ are OR⁶.
 55. The compound of claim 53, wherein R⁶ is acetyl andR⁷ is triflate.
 56. The compound of claim 53, wherein the compound has astructure according to any one of Formulas VI-a to VI-h


57. The compound of claim 53, wherein the compound is selected from


58. A composition comprising a compound of claim 45, and apharmaceutically acceptable carrier.
 59. The composition of claim 58,wherein the pharmaceutically acceptable carrier is water.
 60. A methodof detecting a fungus, comprising: contacting the fungus with one ormore compounds of claim 45, thereby detecting the fungus.
 61. The methodof claim 60, wherein the fungus is an Aspergillus, Candida,Cryptococcus, or Mucormycetes.
 62. The method of claim 60, wherein themethod is an in vivo method of detecting a fungal infection in asubject, and the contacting comprises administering the one or morecompounds to a subject, and the method further comprises subsequentlyperforming diagnostic imaging of the subject, thereby detecting thefungal infection in the subject.
 63. The method of claim 62, wherein thediagnostic imaging of the subject comprises positron emission tomography(PET).
 64. The method of claim 62, wherein the subject is undergoingtreatment of the fungal infection, and the method monitors thetreatment.